FEMS Microbiology Ecology 25 (1998) 205^215
MiniReview
Monitoring the community structure of wastewater treatment
plants: a comparison of old and new techniques
Rudolf Amann a; *, Hilde Lemmer b , Michael Wagner
a
c
ë kologie, Celsiusstr. 1, D-28359 Bremen, Germany
Max-Planck-Institut fuër Marine Mikrobiologie, Arbeitsgruppe Molekulare O
b
Bayerisches Landesamt fuër Wasserwirtschaft, Institut fuër Wasserforschung, Munich, Germany
c
Lehrstuhl fuër Mikrobiologie, Technische Universitaët Muënchen, Munich, Germany
Received 19 September 1997; revised 21 November 1997; accepted 26 November 1997
Abstract
Wastewater treatment is a process of increasing importance in a world with an ever growing human population. Today, most
wastewater treatment processes make use of the natural self-purification capacity of aquatic environments which is the result of
the presence and action of microbial communities. Consequently, wastewater treatment facilities are designed to maintain high
densities and activities of those microorganisms that satisfy the various purification demands. The performance, at least of
large plants, has to be constantly monitored and is subject to strict regulation. Nevertheless, malfunctions resulting in
decreased purification efficacy are frequent. This has, over the decades, prompted many microbiologists to compare the
structure, dynamics and function of these `good' or `bad', but always complex, microbial communities. Part of these studies
was targeted to a basic understanding of the various processes, another part, however, deals with the monitoring of community
structure as a means to direct the plant operation towards higher elimination rates and overall stability. Even though the last
decade has seen a molecular revolution in microbiology, the standard methods for monitoring wastewater treatment plants still
rely on the tools available to the researcher at the beginning of this century, the microscope and agar plates. It is the goal of this
MiniReview to compare the potential of the newly available molecular methods with the old monitoring techniques. z 1998
Published by Elsevier Science B.V. All rights reserved.
Keywords : Wastewater treatment; Monitoring; Community structure ; Microbial ecology; Nucleic acid probe; Microscopy
1. General remarks
In today's world with its increasing human population and the concomitant pollution problems there
is an increasing awareness of the necessity for environmental protection. Our ability to control the pollution caused by human activities is, in the long run,
* Corresponding author.
Tel.: +49 (421) 2028 930; Fax: +49 (421) 2028 790.
crucial for the further development of mankind.
Wastewater treatment is one of the basic processes
in this regard. In former times, the natural ecosystems were, by the so-called self-puri¢cation, able to
deal with the much lower levels of pollution. Microorganisms mineralized the waste and made it available again for primary production. Interestingly
enough, most modern wastewater treatment processes still rely on the action of complex microbial
communities. And even today these communities
0168-6496 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved.
PII S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 0 6 - 3
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R. Amann et al. / FEMS Microbiology Ecology 25 (1998) 205^215
are not deliberately assembled from individual species with known functions, but they remain the result
of natural selection. The art of building an e¤cient
wastewater treatment facility is still to make the best
use of the natural processes, by condensing them in
space and time, or as Curds and Hawkes wrote in
1975 [1]: ``Used water treatment by biological oxidation plants may be regarded as the environmental
control of the activity of the appropriate organisms''.
The group of organisms most directly involved in
wastewater treatment are the bacteria. They dominate, both in numbers and biomass, all other groups
and dominate the processes of mineralization and
elimination of organic and inorganic nutrients.
They are favored, in traditional high load plants
that operate with short sludge retention times, by
their low generation times. Modern low load systems
have high retention times and also allow for the
presence of more slowly growing bacteria and of
organisms with a more complex organization such
as £agellates, amoebae, ciliates or even worms and
insect larvae. The protozoa and metazoa are able to
feed on particulates, such as those coming in with
the sewage or bacterial £ocs. It is generally assumed
that their primary role in the wastewater treatment is
the clari¢cation of the e¥uent.
The microbial communities catalyzing wastewater
treatment have long been viewed as `black boxes'.
Today, even engineers agree that a thorough knowledge of the structure and function of these complex
communities would be a good starting point for future plant optimization. However, with classical
techniques a detailed analysis of the bacterial community structure has not been possible. Therefore,
studies were not performed on those cells that catalyzed reactions of interest, but on so-called indicator
organisms that were morphologically or phenotypically conspicuous and that could be linked, by their
presence, to a good or bad plant performance.
2. Classical techniques for monitoring the microbial
communities of wastewater treatment plants
The study of Smit in 1934 [2] in which he connects
problems in sludge settling to the presence of high
numbers of ¢lamentous bacteria in the sludge is an
early example of a type of wastewater treatment
monitoring that has not changed a lot in the last
six decades. Here, light microscopy was used in an
attempt to visualize the cause of a particular problem. This is, in the case of sludge bulking, straightforward which is in most cases a phenomenon directly linked to the occurrence of ¢lamentous
microorganisms. Since then, the principle approach
has not changed, but it has been considerably re¢ned. Often ¢lamentous bacteria have enough morphological detail like cell dimension, shape or length,
form and branching of ¢laments that they can be
classi¢ed in situ (for details see [3]). This enabled
comparative studies on the presence or absence of
speci¢c ¢laments with regard to sewage composition
and plant operation (e.g. [4^6]). Keys are now available that help in the identi¢cation of ¢laments by the
combination of light microscopy and simple staining
techniques such as those according to Gram and
Neisser. However, it should always be kept in mind
that morphology of all bacteria (including the ¢lamentous types) is a quite unstable character and consequently is not a good foundation for a sound identi¢cation.
Furthermore, eukaryotic organisms are used to
classify activated sludge. Ciliates, rotifers, nematodes
and oligochaetes have enough morphological detail
to be reliably identi¢ed (e.g. [7^9]). Even though
these organisms are not responsible for the primary
degradation activity of the wastewater communities
they contribute to elimination of particulate matter.
Moreover, they are often useful indicators for environmental conditions of the treatment process, e.g.
ammonia-tolerant ciliates such as Tetrahymena indicate high load conditions with preliminary degradation e¤cacy, metazoa such as rotifers and nematodes
indicate long sludge retention times, as found in
sludge deposits or £oated sludge fractions. Based
on the presence or absence of indicator organisms
countermeasures can be suggested in attempts to
change the community structure and function and
thereby the overall plant performance.
It has, however, to be noted that methods based
on simple light microscopic observations do not allow the monitoring of those groups of bacteria that
are responsible for processes such as nitri¢cation,
denitri¢cation or enhanced biological phosphorus removal. Bacteria usually have very limited morphological diversity and this also applies to many of the
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most abundant protozoa, e.g. the nano£agellates and
the small amoebae. Nevertheless, the examination of
sludge by microscopy and with reference to classic
works such as the Microscopic Sludge Investigation
Manual by Eikelboom and van Buijsen [10] or the
Manual on the Causes and Control of Activated
Sludge Bulking and Foaming [11] is today a standard monitoring technique in many plants.
Another classical approach is based on the isolation of bacterial pure cultures, mostly by the agar
plate technique, followed by the identi¢cation and,
if necessary, physiological characterization of the respective strains. For those bacteria that lack a conspicuous morphology this has long been the only
approach to assess their presence and number.
Many of these cultivation studies have been performed, among some well known are those of Prakasam and Dondero [12], Benedict and Carlson [13]
and Kavanaugh and Randall [14]. In the early studies [12,13], media with high carbon concentrations
( s 1 g C l31 ) were applied which select for fast
growing bacteria. Typical examples of such r-strategy saprophytes are the aeromonads. It has long
been realized [12] that by the use of one medium,
even if it is called non-selective, no reasonably complete insight into the community structure can be
obtained. Activated sludge contains a tremendous
diversity of bacteria ranging from extremely slow
growing ones (e.g. the chemolithoautotrophic nitri¢ers) to those with a generation time of less than 30
min (e.g. Aeromonas sp.) and encompassing aerobes
as well as facultative or obligate anaerobes. Many of
these di¡erent bacteria can neither be grown nor
enumerated on a rich medium like a plate count
agar. Various studies have now shown that, even
on optimized media, always less than 20% and usually somewhere between 10 and 1% of those bacteria
visualized by microscopic methods form colonies
[15,16]. This is due in part to the use of the `wrong'
media and cultivation conditions and in part to the
clustering of cells in the activated sludge £ocs. Even
today there is a lack of good methods for converting
activated sludge £ocs or bio¢lms to suspensions of
single cells prior to plating. Therefore, cultivation
based methods are not suitable for exact quanti¢cation of the populations in wastewater treatment.
Scientists interested in quanti¢cation may today
use direct, cultivation-independent methods. These
207
direct methods have also the potential to alleviate
one of the major problems in quantitative population analysis in activated sludge: with cultivationbased methods, even in their improved modern
form (e.g. [17]). This problem is the de¢nitive identi¢cation of a statistically signi¢cant number of colonies, which is very laborious and time-consuming.
This does not mean, though, that there is no place
for pure culture studies in future wastewater microbiology. The characterization of those bacteria, identi¢ed with other methods as important catalysts in
the puri¢cation process, for their physiological potential, can only be performed on pure cultures, even
in the age of molecular biology.
The old cultivation studies have shown that even
when the early scientists isolated the right, important
bacteria for a given plant, they often had problems
in classi¢cation. This was the time of arti¢cial, not
evolutionarily based classi¢cation, and not the enlightened era that we have reached today, largely
based on rRNA-based systematics. As a consequence, recent years have seen major changes in
taxonomy, a process that will likely continue. Strains
that were earlier lumped in the polyphyletic genus
Pseudomonas are now recognized to be distributed
over many species in several subclasses of the class
Proteobacteria and most of the pseudomonads from
environmental sources have been renamed (for a review see [18]).
3. Direct monitoring approaches based on
chemotaxonomy
In the last decade, several attempts have been
made to analyze the bacterial community structure
of activated sludge by direct chemotaxonomic methods. Among these approaches were pro¢les of respiratory quinones [19], polyamine patterns [20] and
phospholipid fatty acid patterns [21]. We will discuss
the principles and potential pitfalls of these approaches by taking as an example the study of Auling and colleagues [20]. After prior analysis of reference strains, diaminopropane (DAP), the main
polyamine compound produced by Acinetobacter
spp., was used as a biomarker for this genus. High
DAP content was found in low load plants with
enhanced
biological
phosphorus
elimination
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1
2
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209
Fig. 1. In situ hybridization of activated sludge from a large municipal plant (Munich-GroMlappen; basin I) with £uorescently labeled,
rRNA-targeted oligonucleotide probes targeting the beta- (Cy3-labeled probe BET42a; red) and gamma-subclass of Proteobacteria (£uorescein-labeled probe GAM42a; green). A fully equipped confocal laser scanning microscope (Zeiss LSM 410; Carl Zeiss, Jena, Germany)
with a 100U oil immersion objective (NA 1.3) was used to create this all-in-focus image. Bar = 10 Wm.
Fig. 2. In situ identi¢cation of the typical zoogloeal ¢ngers in the high load plant of Kraftisried (for details see [33]) by hybridization
with the Cy3-labeled probe ZRA targeting the neotype strain, Z. ramigera ATCC 19544. The all-in-focus confocal laser scanning image
was recorded on a Zeiss LSM 410 with a 40U oil immersion objective (NA 1.25). Bar = 10 Wm.
6
(EBPR) and low DAP content in plants with high
organic loading. Assuming that DAP is the main
polyamine not just of the strains investigated, but
also of all members of the genus Acinetobacter, a
low DAP concentration in the sludge indeed demonstrates that Acinetobacter spp. can not be a major
component of the total community. In contrast, the
interpretation of high DAP levels is not straightforward. DAP might be present in various other bacterial species which have not so far been studied for
their polyamine patterns, e.g. due to problems in
cultivation. Recently, DAP could, for example, be
detected in aeromonads (Kaëmpfer, personal communication) and Aquaspirillum dispar [22]. This might
explain the high DAP content present in EBPR
plants for which a relatively low in situ abundance
of Acinetobacter spp. could be demonstrated by immuno£uorescence [23] and rRNA-targeted probes
[24].
Another general disadvantage of chemotaxonomybased monitoring approaches is the fact that they are
often compromised by varying cellular concentrations of the indicator compounds during the cell
cycle or due to changes in the environment. Therefore, it will be almost impossible to calculate absolute cell numbers from compound concentrations. It
has, however, been pointed out that this type of
monitoring is quick, inexpensive and applicable to
large numbers of samples. It might be a good tool
to follow populations in environments that are well
understood in terms of overall diversity.
4. Identi¢cation of non-conspicuous bacteria by
immuno£uorescence
Early attempts for speci¢c in situ identi¢cation of
individual microbial cells in wastewater treatment
plants were based on immuno£uorescence. This technique was successfully used on various wastewater
bacteria, a study on ammonia-oxidizing bacteria is
discussed here [25]. Detection of this group of
slow-growing bacteria by standard cultivation methods requires incubation times of at least one month.
Detection and enumeration of two serotypes of Nitrosomonas sp. by immuno£uorescent labeling followed by £ow cytometric analysis required just several hours. However, even though this approach can
be directly applied to the complex sample, it still
requires prior isolation of the organism of interest
to produce the antibodies. Other problems are limited di¡usion of the antibody probes through EPS
layers [26] and the potential for antigenic variance
in the target groups.
5. rRNA-based monitoring techniques
Today, the rRNA approach is a powerful tool to
analyze, in a truly cultivation-independent way, the
structure of microbial communities [27]. There have
been two types of rRNA-based studies in wastewater
treatment in the last few years. Group- and genusspeci¢c oligonucleotide probes were used to analyze
directly the community structure of activated sludge
by in situ hybridization ([16,28]; see also Figs. 1 and
2). Usually more than 75% of the bacteria stained
with the general DNA stain DAPI also hybridized
with a bacterial probe. This also proves a high cellular rRNA content which strongly suggests viability
and activity of the majority of cells in wastewater
treatment plants. The use of phylum- and subclassspeci¢c probes, as shown in Fig. 1, for simultaneous
hybridization with probes for the beta- and gammasubclass of Proteobacteria, generally indicated dominance of the beta-subclass Proteobacteria [16].
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Table 1
Occurrence of protozoa and metazoa in activated sludge plants
in Germany
Group
Protozoa
Flagellates
Species
Frequency
Anthophysa sp.
Bodo sp.
Cercomonas sp.
Euglena sp.
Hexamita sp.
Peranema sp.
Trepomonas agilis
Trepomonas rotans
Trigonomonas compressa
Rhynchomonas nasuta
common
frequent
frequent
common
widespread
frequent
widespread
widespread
rare
common
Amoebae
testate amoebae Arcella sp.
Euglypha sp.
Centropyxis sp.
frequent
frequent
frequent
common
Ciliates/Suctoria
Cyrtophorida
Chilodonella uncinata
Odontochlamys alpestris
Pseudochilodonopsis £uviatilis
Trithigmostoma cucullulus
Trithigmostoma steini
Thigmogaster oppositevacuolatus
Trochilia minuta
Gymnostomati- Paradileptus sp.
da
Spathidium sp.
Heterotrichida Metopus sp.
Spirostomum caudatum
Spirostomum teres
Spirostomum minus
Stentor coeruleus
Hymenostomata Cyclidium glaucoma
Cyclidium heptatrichum
Calyptotricha lanuginosa
Cinetochilum margaritaceum
Pseudocohnilembus pusillus
Dexiostoma campylum
Glaucoma scintillans
Paramecium bursaria
Paramecium aurelia complex
Paramecium caudatum
Uronema nigricans
Hypotrichia
Aspidisca cicada
Aspidisca lynceus
Aspidisca turrita
Euplotes a¤nis
Euplotes aediculatus
Tachysoma pellionellum
Oxytricha sp.
frequent
frequent
common
frequent
widespread
frequent
common
rare
rare
rare
rare
rare
rare
rare
frequent
rare
rare
common
widespread
rare
rare
widespread
rare
rare
rare
frequent
frequent
widespread
frequent
common
common
common
Table 1 (continued).
Group
Species
Nassulida
Drepanomonas revoluta
Microthorax pusillus
Peritrichia
Carchesium polypinum
Epistylis chrysemydis
Epistylis entzii
Epistylis plicatilis
Opercularia articulata
Opercularia coarctata
Opercularia nutans
Pseudovorticella monilata
Thuricola kellicottiana
Vorticella convallaria complex
Vorticella aquadulcis complex
Vorticella campanula
Vorticella infusionum complex
Vorticella octava complex
Vorticella microstoma complex
Vorticella picta
Pleurostomatida Amphileptus claparedii
Amphileptus punctatus
Amphileptus pleurosigma
Acineria incurvata
Acineria uncinata
Litonotus alpestris
Litonotus cygnus
Litonotus fusidens
Litonotus lamella
Litonotus crystallinus
Prostomatida
Coleps hirtus
Holophrya discolor
Plagiocampa rouxi
Suctoria
Acineta tuberosa
Tokophrya infusionum
Tokophrya lemnarum
Tokophrya quadripartita
Podophrya sp.
Dendrosoma sp.
Trichophrya sp.
Metazoa
nemathelminthes
rotifera
Cephalodella sp.
Rotaria rotatoria
Philodina sp.
Proales sp.
nematodes
gastrotrichs
tardigrades
plathelminthes
turbellaria
annelids
Aelosoma hemprichi
oligochaetes
Nais sp.
Frequency
widespread
rare
frequent
widespread
frequent
frequent
frequent
common
widespread
widespread
rare
frequent
frequent
widespread
widespread
widespread
rare
rare
widespread
common
rare
rare
frequent
widespread
rare
common
common
widespread
common
common
widespread
common
rare
common
common
widespread
rare
rare
frequent
frequent
widespread
widespread
frequent
widespread
common
rare
rare
rare
Summarized are results of a 2-year survey (1995/1996) of 85 wastewater treatment plants (Lind, personal communication).
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Whereas members of this group were underestimated
by cultivation techniques, members of the gammasubclass of Proteobacteria, most notably the genera
Acinetobacter [24] and Aeromonas [17], were overestimated. Manz et al. [28] used the group-speci¢c
probes to demonstrate di¡erences in the community
structure between municipal and industrial activated
sludge plants. The probes used in these and other
studies were still based mainly on sequences of cultured bacteria.
Other scientists have used direct rRNA sequence
retrieval and subsequent comparative data analysis
of 16S rDNA clone libraries to analyze the microbial
diversity present in activated sludge [29,30]. Whereas
Schuppler et al. [30] focused on clones from the
group of Gram-positive Bacteria with a high DNA
G+C content, Bond et al. [29] partially sequenced
100 statistically selected clones each of two laboratory-scale sequencing batch reactors with strong differences in their phosphate-removing capabilities.
Most clones were a¤liated with the beta-subclass
of Proteobacteria and only few Acinetobacter clones
were found. Both these results were in agreement
with the earlier results of oligonucleotide probing
[16,24]. Bond et al. [29] tried to use the frequencies
of the clones for a comparison of the community
structures of the two reactors. The clone library
from the reactor with the higher phosphate removal
contained more clones related to the genus Rhodocyclus within the beta1-subgroup of Proteobacteria.
However, it has been pointed out before that for
several reasons [27] clone frequencies should not be
regarded as a valid means to estimate abundances of
certain populations. Clearly, the combination of the
two di¡erent rRNA-based techniques, direct rRNA
sequence analysis followed by hybridization with
probes based on the retrieved sequences, would be
the best. However, this is also a very laborious and
time-consuming way to characterize the community
structure of activated sludge at high resolution. Two
such studies were performed recently for the ¢rst
stage of a municipal wastewater treatment facility
receiving a high load of organic waste. In the ¢rst,
simultaneous in situ visualization of up to seven distinct beta-proteobacterial genotypes corroborated
that the high diversity found in the respective clone
library was indeed present in the complex activated
sludge community [31]. The second study resulted in
211
interesting and surprising ¢ndings such as members
of the genus Arcobacter (formerly thermophilic campylobacters), which are epsilon-subclass Proteobacteria, to form quite abundant populations (4% of total
cell counts) in activated sludge [32].
One important advantage of all rRNA-based hybridization techniques is that they are automatically
linked to modern bacterial systematics. A good example is the work of Rossello-Mora and colleagues
[33] on a famous wastewater bacterium, Zoogloea
ramigera. Most strains once designated as Z. ramigera have nothing in common with the neotype
strain, Z. ramigera ATCC 19544. By probing various
activated sludge samples with rRNA-targeted oligonucleotide probes speci¢c for the type strain and two
misclassi¢ed Z. ramigera strains it was demonstrated
that only populations related to the neotype strain
make up a signi¢cant fraction above 1% and, in the
more highly loaded stages sometimes even constitute
more than 10% of the total community. The neotype-speci¢c probe was also the one that visualized
the typical zoogloeal ¢nger-like structures (Fig. 2).
Today, many other bacterial groups important for
wastewater treatment can be tracked by in situ hybridization. For example, probe sets are available for
nitrifying bacteria [34^36] and for ¢lamentous bacteria causing bulking and foaming [37^39].
The monitoring of speci¢c populations in wastewater treatment plants by £uorescent in situ hybridization with rRNA-targeted oligonucleotide probes
can be compromised by high background £uorescence, low cellular rRNA content, or limited probe
permeability of target cells. These problems and approaches to solve them have been discussed in detail
before [27].
Attempts have been made to combine £uorescent
oligonucleotide probing with advanced microscopic
techniques such as confocal laser scanning microscopy for analyzing the spatial distribution of specifically labeled target cells within activated sludge £ocs
and trickling ¢lter bio¢lms [40]. Using this approach
the 3-D arrangements of Gram-negative ¢lamentous
bacteria [37], ammonia-oxidizing bacteria [34] and
Paracoccus sp. [41] were studied. Schramm and colleagues [42] have combined in situ localization of
Nitrosomonas spp. and Nitrobacter spp. with in situ
3
measurements of concentrations of O2 , NO3
2 =NO3 ,
and N2 O using microsensors. They found a good
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match between structure and function, the distribution of the nitrifying bacteria and the zone of nitri¢cation. Such studies are intended to increase our
understanding of the various wastewater treatment
processes. They are, however, too complex to become routine methods in the monitoring of activated
sludge plants in the near future.
The dot blot hybridization technique for quanti¢cation of speci¢c rRNA signature sequences [43] is
yet another modern molecular technique that has
recently been applied to the monitoring of speci¢c
populations in anaerobic wastewater treatment [44].
Here, radioactively labeled, rRNA-targeted oligonucleotides are applied to quantify the relative abundance of a speci¢c rRNA compared to the total
rRNA pool quanti¢ed by a universal probe. The
advantages and disadvantages of this approach are
similar to those discussed for the chemotaxonomic
approach. Dot blot hybridization allows rapid examination of multiple samples in a short time. However, the cellular rRNA content is not stable and
therefore it is di¤cult to extrapolate from relative
fractions to absolute cell numbers. In the case of
rRNA the cellular content is linked to growth rate
for many bacteria and the relative abundance of a
speci¢c rRNA might consequently be a good measurement for the metabolic activity of the respective
population. Based on dot-blot hybridization alone, it
is, however, impossible to attribute changes to £uctuations in cell number or cellular rRNA content.
6. Other hybridization techniques and nucleic acid
based patterns
In contrast to in situ hybridization, dot blot hybridization techniques are already applicable today
to the quanti¢cation of nucleic acids other than
rRNA. Whereas rRNA based techniques allow
only indirect conclusions on the function of a population the detection of a functional gene representative of a de¢ned physiological activity (or its
mRNA) o¡ers more direct evidence for the presence
or abundance of a certain physiological group of
microorganisms and its potential activity. Along
these lines the group of van der Meer (EAWAG,
Switzerland) recently did a thorough study on a continuous culture of Paracoccus denitri¢cans. Induction
and repression of denitri¢cation activity were studied
in a chemostat during changes from aerobic to anaerobic growth conditions [45]. The denitri¢cation activity was monitored by measuring the formation of
denitri¢cation products, individual mRNA levels for
the nitrate, nitrite, and nitrous oxide reductases, and
the concentration of the nitrite reductase enzyme was
assessed with antibodies. On a change from aerobic
to anaerobic respiration, mRNA formation was induced and raised 15- to 45-fold. However, this increase was transient and the mRNA levels declined
within a few hours to levels only slightly higher than
that under aerobic steady-state conditions. Interestingly, the level of nitrite reductase protein increased
slowly during the anaerobic period, reaching a stable
value about 30 h after the switch. Upon a switch
back to aerobic conditions the denitri¢cation
stopped immediately, whereas the levels of mRNA
returned only slowly to their uninduced levels. The
nitrite reductase was not actively degraded and its
decline followed the washout curve. These results
also clearly show that the screening of functional
genes or mRNAs alone is not a straightforward
means to determine in situ activities. Combined studies that use a suite of methods to correlate functional
and structural parameters of wastewater communities are at this time the most promising approach
to gain a more thorough understanding of the important processes in sewage treatment, such as nitri¢cation, denitri¢cation and phosphorus removal.
Last but not least, new techniques should be mentioned here that also have considerable potential for
monitoring population dynamics in complex microbial communities. They essentially all work on nucleic acids extracted from the environment and convert ^ in the ideal case ^ every organism to one
clearly distinguishable band in a pattern. As soon
as the population behind a particular pattern is identi¢ed (e.g. by sequencing the band once), these techniques allow rapid monitoring of multiple samples.
The techniques may target rRNA as well as functional genes. For its sensitivity and speed many of
these techniques are based on PCR [46]. They can
assess di¡erences in the length of restriction fragments of PCR products (e.g. ARDRA [47]) or even
discriminate fragments of identical length but varying sequence by techniques such as DGGE (denaturing gradient gel electrophoresis [48]).
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7. Conclusions
In the last years considerable progress in the analysis of the bacterial part of the biocenoses catalyzing
wastewater treatment has been achieved with new
molecular techniques. However, if we really want
to understand the structure and function of modern
wastewater treatment plants it is important to realize
that these are not any longer the relatively simple
high load systems applied to eliminate carbonaceous
substrate. Today, in the various wastewater treatment plants microbial communities experience, at
certain time intervals, anoxic and/or anaerobic periods. It could be that the more complex modes of
operation in the various types of plant have also
resulted in a higher diversity of community structure.
Furthermore, prolonged biomass retention times allow the development of more slowly growing protozoa and even metazoa. Table 1 gives an overview of
the di¡erent species that have been identi¢ed as regular inhabitants of activated sludge plants in Germany (Lind, personal communication). Even though
protozoa and metazoa already serve as indicator organisms we are far from an understanding of their
impact on community structure. Intuitively, most microbiologists and engineers dealing with the `living
part of wastewater treatment' still regard the bacterial community structure as mainly resulting from
the physico-chemical environment, and neglect the
potential impact of grazing. In the ¢eld of microbial
ecology the discussion on the relative impact of `bottom-up' (e.g. nutrient availability) vs. `top-down'
(e.g. grazing by protozoa) regulation on the actual
community structure has been most fruitful. It is
overdue that environmental biotechnologists start
to consider the potential e¡ects of microbial food
webs. Along these lines, sludge scumming, a nuisance
observed in many of the modern nutrient removal
plants and frequently linked to high abundance of
hydrophobic, Gram-positive ¢lamentous bacteria,
might be explained in the future by top-down regulation, resulting from the formation of grazing resistant forms. From lake systems it is well known that
protozoan and metazoan grazing can cause shifts in
bacterial community structures towards ¢lamentous
forms [49]. The integration of higher trophic levels
into our models of wastewater treatment might indeed be the key to a better understanding of struc-
213
ture-function correlations. Molecular techniques,
such as those described in this MiniReview for
studying bacteria, might also be highly useful for
the enumeration and identi¢cation at least of the
smaller protozoa [50]. A thorough description of
the microbial community ^ including the Eucarya
in addition to the Bacteria ^ could help in the future
to elucidate adaptation and selection mechanisms in
the various trophic levels. This type of basic biological understanding could indeed support the theory
and practice of wastewater treatment.
Acknowledgments
R.A. gratefully acknowledges ¢nancial support
from the Deutsche Forschungsgemeinschaft (Am
73/2), the Freistaat Bayern (FORBIOSICH, FORGEN) and the European Community (HRAMI projects). Excellent technical assistance was provided by
Mrs. Sibylle Schadhauser. We wish to thank George
Lind for the data of her study on protozoa and metazoa in wastewater treatment plants. Without the
support of Dr. Wolfgang Ludwig and Prof. Dr.
Karl-Heinz Schleifer this work would not have
been possible.
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